Vascular magnetic imaging method and agent comprising biodegradeable superparamagnetic metal oxides

ABSTRACT

The preparation and isolation of biodegradable superparamagnetic MR imaging contrast agents for the vascular compartment is described. These aggregates are comprised of individual biodegradable superparamagnetic metal oxide crystals which aggregates have an overall mean diameter less than about 4000 angstroms. The preferred vascular imaging contrast agent is comprised of aggregates of iron oxide crystals having an overall mean diameter less than about 500 angstroms. These contrast agents may be associated with a macromolecular species, which assist, among other things, in the preparation of these extremely small materials, and may be dispersed or dissolved in a physiologically acceptable medium. Preferred media also stabilize the materials against further aggregation even under harsh sterilization conditions. The autoclaved biodegradable superparamagnetic iron oxides of the invention are ideally suited for a pharmaceutical preparation and enjoy several advantages over prior intravascular imaging contrast media including low osmolality, low effective dose requirements, high relaxivities, long blood lifetimes, rapid biodegradability, and versatility with respect to a wide range of applicable MR data acquisition parameters.

CROSS REF. TO RELATED APPLICATIONS

This application is a continuation-in-part of applicant' priorco-pending U.S. application Ser. No. 067,586 filed June 26, 1987, nowU.S. Pat. No. 4,827,945, which is, in turn, a continuation-in-part ofthe applicants' prior co-pending U.S. application Ser. No. 882,044 filedJuly 3, 1986, now U.S. Pat. No. 4,770,183, the disclosures of which areincorporated herein by reference.

1. INTRODUCTION

This invention relates to methods for enhancing magnetic resonance (MR)images of the vascular compartment of animal or human subjects. Themethods of the present invention involve the use of biodegradablesuperparamagnetic contrast agents which enhance MR images of thevascular compartment. These methods, in turn, allow one to image organor tissue perfusion, as well as blood flow. The use of these contrastagents, preferably administered as a superparamagnetic fluid, offerssignificant advantages over existing methodologies including inter aliahigh proton relaxivities, rapid biodegradability, control of bloodlifetimes, versatility in choice of pulse sequence weighting schemes andother MR experimental parameters, low dosage requirements, lowosmolality, and little or no toxicity.

2. BACKGROUND OF THE INVENTION

Magnetic resonance (MR) imaging is widely regarded as a powerfultechnique for probing, discovering, and diagnosing the presence andprogress of a pathological condition or disease. The MR method ofimaging is also regarded as the least invasive of the imaging techniquespresently available and does not expose the patient or subject topotentially harmful high-energy radiation, such as X-rays, orradioactive isotopes, such as technetium-99m. Technological developmentsin both instrumentation and computer software continue to improve theavailability and quality of the images produced. Researchers discoveredquickly, however, that the relative differences between the chemical andmagnetic environments of water molecules, whose proton nuclei provide byfar the largest source of measurable signal intensity within the body,whether these molecules be located in organs, tissues, tumors, or in thevascular compartment, are often quite small and, consequently, theresulting images are poorly resolved. Fortunately, this inherentlimitation can be overcome by the use of proton relaxation agents, alsoknown as contrast agents, which are absorbed selectively by differenttypes of tissues and/or sets of organs, and thus create a temporarycondition in which the magnetic environments of neighboring watermolecules are measurably dissimilar.

According to their magnetic properties, there are three general types ofMR contrast agents: paramagnetic, ferromagnetic, and superparamagnetic.The weak magnetism of paramagnetic substances arises from the individualunpaired electrons while the stronger magnetism of ferromagnetic andsuperparamagnetic materials results from the coupling of unpairedelectrons made possible by their presence in crystalline lattices.Ferromagnetic materials retain their magnetism in the absence of anapplied magnetic field while superparamagnetic materials lose theirmagnetism when the applied magnetic field is removed. With respect toeffects on proton relaxation, paramagnetic agents have been termed T₁type agents because of their ability to enhance spin-lattice orlongitudinal relaxation of proton nuclei. Ferromagnetic agents have beentermed T₂ type agents because of their specific effects on T₂, sometimescalled the spin-spin or transverse relaxation.

There are three major disadvantages to the use of paramagnetic chelatesas vascular MR contrast agents. The first is that many low molecularweight, ionic materials which are commonly used as paramagnetic chelatesare hypertonic. The use of hypertonic solutions often results in adversereactions upon injection. The second disadvantage is that paramagneticchelates have short blood lifetimes whereas a vascular MR contrast agentshould remain confined to the vascular compartment for long periods oftime. Third, removal of paramagnetic chelates from the vascularcompartment can result in release of the paramagnetic ion from thechelate. Paramagnetic ions of iron, manganese, and gadolinium, forexample, are toxic in their free ionic form. Vascular MR contrast agentsshould have a benign metabolic fate after removal from the vascularcompartment. These three disadvantages are explained further below.

Paramagnetic materials that have been used as T₁ contrast agentsgenerally include organic free radicals as well as transition metalsalts and chelates. These compounds can be quite soluble and, in thecase of most transition metal complexes, are highly charged, ionicspecies. Due to their relatively low relaxivities (their ability toincrease the relaxation rates of protons as a function of dose), highconcentrations of transition metal chelates are needed to effect usefulalterations in the relaxation times of blood. In addition, the ionicnature of many transition metal ion salts and chelates contributes tothe high osmotic pressure of the injected diagnostic solutions. The endresult is that solutions of paramagnetic materials, whether they areused as MR agents or not, tend to be hyperosmotic relative to blood. Theadministration of hyperosmotic solutions into the subject is widelybelieved to be a major cause of adverse reactions to radiographic and MRcontrast media (See, McLennan, B. L. Diagnostic Imaging Supplement 1987,16-18 (December); "Contrast Media: Biological Effects and ClinicalApplication," Vol. I, Parvez, Z., Moncada, R., and Sovak, M. (Eds.), CRCPress, Boca Raton, Fla. (1987)).

The usual approach to the development of MR contrast agents confined tothe vascular compartment for long periods of time is to increase themolecular weight of paramagnetic chelates by attaching the chelates tohigh molecular weight polymers. After injection, high molecular weightforms of the chelates cannot be excreted by glomerular filtration, andconsequently have longer residence times within the vascularcompartment. High molecular weight forms of chelates can be made bycovalently attaching chelators to macromolecules such as human serumalbumin (Schmiedl et al. Radiology 1987, 162, 205-210)). With thisapproach to the design of vascular MR contrast agents, the fate of thegadolinium after degradation of the agent presents serious problems. Thelong term retention of gadolinium not eliminated by glomerularfiltration, and the potential for delayed toxicity from that element,pose major obstacles to the administration of high molecular weightgadolinium chelates to humans.

A major disadvantage of present ferromagnetic contrast agents is thatsuch materials are relatively large and, frankly particulate incharacter. Frankly particulate materials, those generally having overalldimensions between about 0.5-10 microns, are quickly removed from theblood by the phagocytic action of the cells of the reticuloendothelialsystem, limiting the duration of their effects on the spin-spin andspin-lattice relaxation times of blood. Hence their usefulness asvascular MR contrast agents is limited. All particulate agents suffer asimilar limitation.

3. DEFINITIONS

Unless otherwise noted, the term "sterilized" describes a sample or apreparation which has been subjected to any method known in the artwhich completely destroys all bacteria and other infectious agents whichmay be present in the sample or preparation. Nonlimiting examples ofsuch methods include autoclaving, ultraviolet or gamma irradiation, coldmembrane filtration, or chemical treatment. The resulting preparation isthen suitable for in vivo and/or clinical use with or without furthertreatment. The term "biodegradable" describes a property of a compoundor complex which allows the compound or complex to be broken down intosmaller innocuous components and be excreted from or utilized within thebody. The term "polyfunctional" applies to a molecule which contains aplurality of identical functional groups. A "macromolecular species" mayinclude any molecule, natural or synthetic, which has a molecular weightin excess of 1 kilodalton.

Abbreviations used in the text are defined below.

T₁ =spin-lattice relaxation time

T₂ =spin-spin relaxation time

EM=transmission electron microscopy

MR=magnetic resonance or nuclear magnetic resonance (NMR)

RES=reticuloendothelial system

MW=molecular weight

kD=kilodalton

4. SUMMARY OF THE INVENTION

This relates to a method for enhancing an MR image of the vascularcompartment of an animal or human subject. The enhanced MR image isrealized by administering to such an animal or human subject aneffective amount of a contrast agent prepared from aggregates ofindividual biodegradable superparamagnetic metal oxide crystals. Theaggregates, whose metal oxide crystals may be associated with amacromolecular species, are dispersed in a physiologically acceptablemedium, preferably an aqueous citrate buffer, which stabilizes theaggregates of the resulting fluid against further aggregation even underthe sometimes extreme conditions of sterilization. The preparation ofbiodegradable superparamagnetic aggregates are described whoseproperties and characteristics, such as overall sizes and bloodcirculation lifetimes, for example, may be varied.

One of the objects of the present invention is to provide apharmaceutical preparation of a biodegradable superparamagnetic contrastagent suitable for human clinical use in magnetic resonance experiments.In particular, preferred embodiments of the invention have great utilityin obtaining enhanced images of the vascular system, of the blood pool,or of organ or tissue perfusion. By practicing the methods of theinvention, blood vessels, arteries, veins, capillaries, as well as lymphvessels may be imaged successfully. In particular capillary beds ofvarious organs or tissues such as the brain, the kidney, the lung, andthe heart can be visualized. Such methods can thus provide valuableinformation regarding the condition of these vessels, organs, or tissuesincluding the presence of microocclusions, embolisms, aneurysm,restricted blood flow, and the onset or recession of arterial disease.

It is also an object of this invention to provide a contrast agent whichcan serve as a brightening or a darkening agent, or both, and which iseffective at low doses. This invention also seeks to provide a contrastagent which is rapidly biodegraded, being either excreted from orutilized within the body of the subject, as evidenced by a return of theproton relaxation rates of affected tissue to pre-administration levelswithin about one week of the initial administration. Yet another objectof the present invention is to provide sterile solutions of a contrastagent which have a low osmotic pressure, low enough, in fact, that addedsalt may be included in the formulation if so desired. Hypotonicsolutions of contrast media may thus be prepared, if so desired. It isalso an object of the present invention to provide a contrast agentwhose blood lifetime may be adjusted to accommodate the variable needsof the MR imaging experiment.

It is thus an object of the instant invention to provide an effectivecontrast agent with all the advantages enumerated above yet issubstantially free of the limitations and objectionable aspectsassociated with paramagnetic and ferromagnetic materials.

After a thorough consideration of the foregoing disclosures and thosethat follow, other objects of the present invention become readilyapparent to those readers skilled in the art.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chromatogram obtained from a Sepharose B gel exclusionfractionation of a biodegradable superparamagnetic fluid.

FIG. 2 is a plot of the blood spin-lattice relaxation rate versus timefor unfractionated and fractionated superparamagnetic material.

FIG. 3 is an electron micrograph of a sterilized superparamagnetic fluidprepared according to the standard methods of the invention.

FIG. 4 is a plot of the "brightening" effect of a number of contrastagents as a function of their solution concentration.

6. DETAILED DESCRIPTION OF THE INVENTION

Superparamagnetic metal oxides are a unique class of magnetic materialsbecause of their large effects on both the T₁ and T₂ relaxation times ofmagnetically active nuclei. We have described in detail the utility ofbiodegradable superparamagnetic materials as MR contrast agents in priorco-pending U.S. application Ser. Nos. 882,044 and 067,586, filed July 3,1986 and June 26, 1987, respectively. The disclosures in bothApplications are incorporated herein by reference. Others have describedthe use of superparamagnetic contrast agents since (See, for example,U.S. Pat. No. 4,675,173 issued to Widder; Hemmingson et al. Acta.Radiologica 1987, 28(6), 703; Saini et. al Radiology 1987, 162, 211).When present as a component of a superparamagnetic lattice, transitionmetals, such as iron, are more potent enhancers of proton relaxationthan the same metal as the central ion of a simple paramagnetic chelate.This increased "potency" of the superparamagnetic iron is reflected in,for example, the spin lattice relaxivity value measured for abiodegradable superparamagnetic fluid, comprising the biodegradablesuperparamagnetic iron oxide and a citrate buffer, which value is 50times greater than that found for a chelated form of iron. Indeed, asthe sizes of the aggregates of iron oxide crystals decrease, theireffects on T₁ increase relative to their effects on T₂. As disclosed inthe present invention, superparamagnetic fluids comprised of very smallmetal oxide aggregates are excellent intravascular MR contrast agentsand can be used in a variety of modes including T₁ -weighted(brightening agents), T₂ -weighted (darkening agents), or combinationsof these and other pulse techniques. The present invention is directedto agents and to a method for the enhancement of MR images of thevascular compartment. The enhancement of the MR image is made possibleby the use of a sterile biodegradable superparamagnetic contrast agentwhose magnetic component is comprised of three-dimensional aggregate ofindividual biodegradable superparamagnetic metal oxide crystals.

An important prerequisite for the successful use of biodegradablesuperparamagnetic materials as intravascular agents in humans is theability to control the blood lifetimes of the materials. In other words,an effective intravascular agent must have a useful lifetime in thecirculatory system so that the MR experiment may be completed before asignificant proportion of the administered contrast agent is removedfrom circulation by organs or tissues of, for example, thereticuloendothelial system (RES). The injection of higher concentrationsof any contrast agent will, of course, increase its longevity in thevascular compartment and prolong its effect on the nuclear relaxationtimes of the blood. Such a strategy, while useful in demonstrating theefficacy of an MR contrast agent in animal models of human pathology, isnot ideal for the formulation of a pharmaceutical, clinical form of thecontrast agent. Safety considerations preclude the use of overly largedoses of a pharmaceutical or diagnostic agent. Imaging techniques, suchas those described herein, which are able to enhance the blood poolwhile maintaining the minimum effective dose are highly desirable.Moreover, the ability of the practitioner to manipulate or control theblood lifetime of the biodegradable superparamagnetic contrast agents ofthe invention permits an unsurpassed flexibility previously unavailablein the design and operation of the MR experiment. A variety ofpharmacokinetic patterns might be used, for example. Hence, for anexperiment designed to delineate tissue blood pool volume, an agent witha long blood lifetime would be used having all the advantages outlinedabove for biodegradable superparamagnetic materials. In otherapplications, an agent which is rapidly removed from the vascularcompartment may be used to "wash in" to damaged tissue and an imagetaken very soon thereafter. Areas where the blood brain barrier havebroken down due to the presence of tumors may be successfully examinedusing these methods. Moreover, because discrete size ranges of thecontrast agent can be obtained, for example, by size exclusionchromatography, the practitioner can tailor and control the amount oftime (e.g., blood halflives) that these agents spend circulating withinthe vascular compartment. Generally, the blood lifetime is inverselyproportional to the overall size of the contrast agent. Some agents ofthe present invention, because of their extremely small dimensions, areable to circulate within the vascular compartment for relatively longperiods of time. The crystalline nature of these agents, which agentscharacteristically provide solutions of low osmolality compared withparamagnetic agents, in combination with the above-mentioned propertiesand characteristics make these biodegradable superparamagnetic metaloxides uniquely suitable as intravascular contrast agents.

The biodegradable superparamagnetic metal oxides can be prepared as asuperparamagnetic dispersion or as a superparamagnetic fluid by generalmethods described previously in applicants, prior co-pending U.S.Applications identified above. These procedures include precipitatingthe metal oxide crystals, in the presence or absence of a macromolecularor polymeric species, from a solution of the trivalent and divalent ionsof a metal by the addition of a sufficient quantity of base. Preferredmetal ions include those of chromium, cobalt, copper, iron, manganese,molybdenum, nickel, and tungsten. A solution of hydrated ferric andferrous salts is particularly preferred. The alkaline reaction may becarried out in the presence of a macromolecular species which aids indispersing the resultant superparamagnetic metal oxide crystals. Ingeneral, the aggregates that form initially, in the absence of thepolymeric substance, are larger. Some suitable macromolecular speciesinclude proteins, polypeptides, carbohydrates, mono-, oligo-, orpolysaccharides. Preferred macromolecules include human or bovine serumalbumin, polyglutamate, polylysine, heparin, hydroxyethyl starch,gelatin, or dextran, with the lattermost being particularly preferred.The dark slurry which forms after the addition of base is then sonicatedto further reduce the size of the aggregates. The sonication also servesto oxygenate the mixture and assures the full oxidation of the divalentmetal ion to the trivalent oxidation state. Larger particulateaggregates that may form during the procedure may be removed bycentrifugation. The resulting supernatant contains onlynonparticulate-sized aggregates.

The next stage of the preparation involves the dilution of thesupernatant with distilled water and subsequent ultrafiltration througha hollow fiber dialysis apparatus. The diluted supernatant is dialysedagainst an aqueous solution or buffer of a polyfunctional organicmolecule. Molecules containing positively charged groups may beutilized. These molecules may include but are not limited to polylysine,polyornithine, and polyarginine. However, the functional groups of theorganic molecule are preferably ionizable to give negatively chargedgroups such as phosphates, phosphinates, phosphonates, sulfinates,sulfonates, carboxylates, and the like. Polycarboxylate compounds areparticularly effective, with salts of citrate ion being most preferred.Besides being commonly used and widely accepted as safe in clinicalpreparations, the aqueous citrate buffer serves to stabilize theaggregates of the resulting superparamagnetic fluid to furtherclustering or aggregation. The inventors have found, quite unexpectedly,that the resulting fluid is stable even under the extreme conditions ofprolonged heating in an autoclave. In addition, the dialysis step alsoremoves most of the macromolecular species, e.g. dextran. Also, dialysisagainst citrate scavengers isolated free metal ions probably throughcoordination with the anionic groups of the polyfunctional molecule. Ifso desired, greater than ninety percent of the dextran used initiallycan be removed in this manner.

The resulting dialysed superparamagnetic fluid is then diluted furtherand sterilized by any means well known in the art. Sterilization may beaccomplished by autoclaving, irradiation, sterile filtration, chemicaltreatment, or other means. Sealing the samples in appropriate containersand heating them in an autoclave is most convenient. It is understoodthat, depending upon the particular application at hand, substantiallyall the added macromolecular species may be removed or anotherintroduced by any appropriate means well known in the art includingexhaustive dialysis.

The size distribution and architecture of the metal oxide crystalaggregates can be examined by light scattering methods and by electronmicroscopy (EM). The EM studies are particularly revealing and show thatthe aggregates are indeed comprised of individual metal oxide crystalswhich are interconnected to form irregularly shaped three-dimensionalstructures (See, for example, FIG. 3). The dimensions of the aggregatesdepend on the number of component crystals present and, for one specificexample, fall in the range of 10-125 nm. The majority of the aggregateshave dimensions smaller than about 50 nm. The results of lightscattering methods give similar, but slightly larger, values. Thisslight difference is probably due to the fact that larger particles willtend to scatter more light and thus skew the value slightly toward thehigher end. Light scattering methods give a value of about 75 nm for theoverall mean diameter of the sample described above.

In a specially modified procedure, extremely small superparamagneticmaterials particles are obtained at the outset. In this modification, asolution of ferric and ferrous salts is adjusted first to a pH of about2.3 before the addition of dextran. The resulting mixture is stirred andheated to about 60°-70° C. for several minutes and then allowed to coolto a temperature of about 40°-45° C. To this solution is added asufficient amount of ammonium hydroxide to bring the pH to about 10. Theresulting suspension is then heated to about 100° C. until a blacksuspension forms. After an ultrafiltration step to remove most of theunbound dextran, light scattering experiments showed that dextranizedparticles of this preparation have an overall mean diameter of about 40nm.

The superparamagnetic fluid prepared according to the general methodscan be separated into fractions of decreasing aggregate diameter by sizeexclusion gel chromatography with a suitable buffer as eluent. Thecollected fractions are analyzed by UV-vis spectroscopy for metal oxidecontent and by light scattering methods for an estimate of their crystalaggregate dimensions. Predictably, the larger aggregates elute firstfollowed by the smaller ones. The fractions can be divided into fourgroups, for the sake of convenience, and tested separately. A standardsolution comprised of dextran blue (2,000 kD), ferritin (443 kD), andbovine serum albumin (65 kD) was fractionated under the same conditionsas the superparamagnetic fluid. The fraction number or elution volume atwhich the known proteins emerged served as points of reference fromwhich the average "molecular weights" of the crystal aggregates of thecontrast agent fractions could be related. The results are summarized inSection 7.5.

Proton relaxation experiments are carried out in vivo by intravascularlyinjecting mice with solutions of fractionated as well as unfractionatedcontrast agents. The effect on proton relaxation, T₁ for example, may bemonitored as a function of time for each of the samples, and the resultsshow that the smaller the aggregate size the longer the blood serumlifetime of the contrast agent. The unfractionated superparamagneticcontrast agent shows a moderate serum blood halflife, Blood t_(1/2), ofabout 13.5 minutes while the smallest aggregates have a Blood t_(1/2)value of 49.6 minutes.

These superparamagnetic metal oxide preparations serve as excellentcontrast agents for MR imaging. They may be successfully used asbrightening agents, for example, as can be demonstrated by a relativecomparison of the Brightness/Intensity effects of the materials of theinvention versus samples of pure water and aqueous solutions of typicalparamagnetic T₁ relaxation agents such as manganese(II) chloride orferric chloride. All the superparamagnetic metal oxide preparationsdescribed above are able to brighten water signals relative to purewater and in one case, the brightness effects even exceeds that ofmanganese(II) chloride, heretofore recognized as a very effective T₁contrast agent (See FIG. 4).

7. EXAMPLES 7.1. PREPARATION OF STERILIZED SUPERPARAMAGNETIC FLUIDS

To a vigorously stirred aqueous 16% ammonium hydroxide solution (5liters) of dextran (2.500 kg, 10-15 kilodalton) is added gradually, andover a 5 minute period, an aqueous solution (5 liters) of ferricchloride hexahydrate (FeCl₃.6H₂ O, 0.755 kg) and ferrous chloridetetrahydrate (FeCl₂.4H₂ O, 0.320 kg). The black magnetic slurry whichforms is then pumped at a rate of about 0.4 liters per minute through acontinuous flow sonicating apparatus. It is sometimes advantageous,although not necessary, to heat the mixture through a sonicatingapparatus which comprises a sonicator connected in series to a 100° C.heating coil unit followed by a cooling coil unit. The resultingdispersed mixture is next centrifuged at 2,000×g for 20 minutes toseparate the larger aggregates which are discarded.

The supernatant is diluted with deionized, sterile water to a volume ofabout 20 liters. Ultrafiltration of the diluted supernatant againstwater and citrate buffer is then carried out in a noncontinuous fashionusing a large hollow fiber dialysis/concentrator, Model DC 10 (AmiconCorp., Danvers, Mass.), equipped with a 100 kilodalton molecular weightcutoff dialysis cartridge. In this manner, a substantial amount of thedextran used initially is removed along with any free metal ions.

After ultrafiltration, a solution of 0.20M Fe and 0.025M citrate isobtained by the addition of 1M citrate and adjusting to a pH of about 8with 1N NaOH. The solution is then autoclaved for 30 minutes (121° C.).Autoclaving is the preferred technique for sterilization since thebottle or container need not be sterile prior to fill. An alternative tosterilization is filtration, but superparamagnetic fluids at highconcentrations filter poorly. Dilute superparamagnetic fluids can befilter sterilized, but the added water must then be removed understerile conditions.

7.2 REMOVAL OF DEXTRAN FROM STERILIZED SUPERPARAMAGNETIC FLUIDS

The dextran remaining after ultrafiltration is largely dissociated fromthe superparamagnetic iron oxide-dextran by autoclaving.

To determine what part of the dextran is attached to superparamagneticiron oxide after autoclaving, dialysis is used to separate dextran fromsuperparamagnetic iron oxide-dextran complexes. The autoclaved 0.20M Fesolution from above is diluted into 25 mM citrate buffer (pH 8) to aniron concentration of about 2 mM and the solution incubated overnight at37° C. to release any dextran weakly adsorbed to the superparamagneticiron oxide. The solution is then applied to a centrifuge micropartitionsystem equipped with a cellulose acetate membrane (Amicon, Danvers,Mass.), where the filtrate is forced through the membrane bycentrifugation. The dextran employed initially has a molecular weight of10-15 kD and passes through the membrane while superparamagnetic ironoxide does not.

Prior to autoclaving (but after ultrafiltration) there were 8.45 mg/mLof dextran present in a solution containing 11.2 mg/mL of Fe (0.2MFe=11.2 mg Fe/mL). The concentration of dextran passing through themicropartition system membrane was 7.49 mg/mL, indicating that most ofthe dextran had detached from the superparamagnetic iron oxide by theautoclaving step. Iron is measured by atomic absorptionspectrophotometry after dissolving the iron oxide in 0.01N HCl. Dextranis measured by a phenol-sulfuric acid method for total carbohydrates(See, C. E. Meloan and Y. Pomeranz, "Food Analysis LaboratoryExperiments," The Avi Publishing Co., Westport Conn., pp. 85-86 (1973)).

The small aggregates of superparamagnetic iron oxide crystals seen in EMstudies are largely uncoated after autoclaving and are stabilizedthrough their interactions with polyvalent anions such as citrate. Themajority of the dextran is present as a dialyzable species free insolution.

7.3 MODIFIED PROCEDURE FOR THE PREPARATION OF EXTREMELY SMALLBIODEGRADABLE SUPERPARAMAGNETIC AGGREGATES

To an aqueous solution (250 mL) of FeCl₃.6H₂ O (35 g) and FeCl₂.4H₂ O(16 g) is added a sufficient amount of aqueous 10% sodium carbonate tobring the pH of the solution to a value of about 2.3. Solid dextran (150g) is then added. The solution is stirred and heated to about 60°-70° C.for about 15 min and then allowed to cool to 40°-45° C. To the reddishsolution is added aqueous 7.5% NH₄ OH to a final pH between 9.5 and10.0. A greenish suspension is produced which is subsequently heated to95°-100° C. for 15 min. The resulting black suspension is then subjectedto an ultrafiltration step using an Amicon RA 2000 hollow fiber dialysisunit equipped with a cartridge having a nominal cutoff of 100kilodaltons. Light scattering measurements reveal that the dextranizedparticle has an overall mean diameter of about 40 nm.

7.4 GEL EXCLUSION CHROMATOGRAPHY OF SUPERPARAMAGNETIC MATERIALS

The superparamagnetic fluid described in Section 7.1 can be applied tothe top of a chromatography column (2.5 cm×100 cm) packed with Sepharose4B. The sample is eluted with 20 mM sodium citrate buffer (pH=8).Fractions are collected and analyzed for their optical density at 340nm. A separate sample containing a mixture of known proteins is treatedand fractionated under the same chromatography conditions. A plot of theoptical density (OD) versus the fraction number and elution volumeyields a chromatogram which is illustrated in FIG. 1. The referenceproteins elute at the fraction numbers indicated at the bottom of theplot. The overall mean diameter in nanometers may be determined for agiven set of superparamagnetic fluid fractions by light scatteringmethods. These results are listed across the top of FIG. 1. As isreadily evident from the chromatogram and plot, the overall sizedecreases with increasing fraction number and elution time. Likewise,the higher molecular weight proteins elute faster than the smaller ones.The eluted fractions of superparamagnetic materials can be combined asfollows: Sample A, fractions 29-31; Sample B, fractions 34-37; Sample C,fractions 43-49; and Sample D, fractions 55-62.

7.5 DETERMINATION OF BLOOD LIFETIMES OF SUPERPARAMAGNETIC MATERIALS AS AFUNCTION OF SIZE

The samples B, C, and D, described above, are intravenously injectedseparately into a rat at the same dose of 2 mg of iron per kg of rat.The spin-lattice relaxation rate, 1/T₁, can then be measured as afunction of time and reflects the blood concentration. FIG. 2 is agraphical representation of the results for samples B, C, D, andunfractionated superparamagnetic fluid. An examination of the plot inFIG. 2 shows that the blood lifetimes of the different fractionsincrease with decreasing overall size. Table I summarizes the results ofthe above experiments. The blood half-life (Blood t_(1/2)) is determinedfrom a nonlinear least squares fit to a single exponential decayprocess. The molecular weight (MW) values of samples A, B, C, and D arethose estimated from extrapolated values derived from the standardproteins.

                  TABLE I                                                         ______________________________________                                        PROPERTIES OF NATIVE AND FRACTIONATED                                         SUPERPARAMAGNETIC FLUID                                                       Sample Fraction #                                                                              OMD.sup.a                                                                              MW     Blood t.sub.1/2                                                                      Dose                                  ______________________________________                                        Native.sup.b                                                                         All        75 nm   -kD    13.5 min                                                                             1 mg/kg                               A      29-31      97      1,500  --     2                                     B      34-37      46      500     4.05  2                                     C      43-49      18      170    16.6   2                                     D      55-62     <15       60    49.6   2                                     ______________________________________                                         .sup.a Overall mean diameter as measured by light scattering methods.         .sup.b Unfractionated fluid prepared by the general methods.             

7.6 TRANSMISSION ELECTRON MICROSCOPY STUDIES

An electron micrograph of the sterilized superparamagnetic fluid,prepared according to 7.1 is shown in FIG. 3. The EM study shows theindividual particle to be aggregates of iron oxide, with individual ironoxide crystals having a diameter of about 5-7 nm. The crystals of eachparticle are in direct contact with each other and are not separated by,or embedded in, a matrix or polymer of any kind. EM studies (beyondthose shown in FIG. 3) have indicated the presence of aggregates from assmall as 10 nm to as large as 300 nm. Both EM and gel chromatography(see Section 7.4) indicate that the superparamagnetic fluid is aheterogeneous mixture of different-sized particles. EM studies revealthe presence of a large number of small aggregates which comprise a verysmall percentage of the volume of iron mass. Consequently the averagevolume of an aggregate by EM studies is only about 20 nm. (Averagevolume =the total volume of all aggregates divided by the total numberof aggregates.)

For EM studies it is important to prevent changes in size distributionof particles during sample preparation. The preparation of the samplefor EM studies is as follows. A solution of 5% molten agar is added tothe sterile superparamagnetic fluid to give a final agar concentrationof 1% and the agar allowed to harden. The agar is minced into pieces ofabout a millimeter and dehydrated by addition of ethanol/water mixturesof decreasing water content until water is replaced by 100% ethanol. Theethanol is then replaced by propylene oxide in an analogous fashion. Thepropylene resin finally yielding a 100% epoxy resin which is hardened bycuring overnight at 60° C. The hardened resin is then sliced with anultramicrotone (0.5 micron thick sections) and placed on a 400 meshcopper grid.

A Phillips model 410 LS transmission electron microscope is used to takemicrographs at 100 kV.

7.7 SUPERPARAMAGNETIC FLUIDS AS BRIGHTENING AGENTS

Superparamagnetic materials are used as MR contrast agents due to theirability to promote the relaxation of magnetically active nuclei. As theparticle size of iron oxide cluster in superparamagnetic fluids becomessmaller and smaller, they become more powerful as brightening agents inT₁ -weighted MR pulse sequences. FIG. 4 shows the effects of differentconcentrations of four MR contrast agents on the signal intensity (S) ofan MR image using a T₁ -weighted pulse sequence of TE=200 msec and TR=15msec. The imager was a GE CSI 2 Tesla imager. The signal intensity ofdistilled water is set at 1.0.

Various concentrations of agents were added to distilled water and themolar concentration of metal is given on the x-axis. Agents thatbrighten increase the value of S while those that darken decrease thevalue of S. A standard T₁ type MR brightening agent is MnCl₂ and has astrong image brightening effect over a wide range of concentrations. Astandard, more purely T₂ type MR agent is provided by a large silanizedcluster of superparamagnetic materials made according to the teachingsof U.S. Pat. No. 4,695,392 which is incorporated herein by reference. Ona molar basis the smallest fraction obtained from the chromatogram ofFIG. 1 (D of Table I) is a more potent brightening agent than MnCl₂,while the larger parent material (Native of Table I) is considerablyless potent as a brightening agent. As shown in FIG. 4, the largesilanized cluster is the least potent T₁ type agent. The strong effectsof sterile superparamagnetic fluids on MR image brightening,particularly as particle size decreases, allows such materials to beused as vascular MR imaging with T₁ - or T₂ - weighted pulse sequences.When T₁ -weighted sequences are used, such materials perform as contrastagents in a manner analogous to paramagnetic brightening agents such asGd/DTPA or MnCl₂.

It should be apparent that other modifications and embodiments can becontemplated without departing significantly from the scope and spiritof the present invention. The invention should, therefore, not belimited by the foregoing examples and descriptions thereof but only asenumerated in the following claims.

What is claimed is:
 1. A biodegradable superparamagnetic metal oxidecomprising aggregates of individual biodegradable superparamagnetichydrated metal oxide crystals, said metal oxide (i) having an individualcrystal diameter of about 500 angstroms or less and aggregates with anoverall mean diameter of about 4000 angstroms or less, as measured bylight scattering methods; (ii) having a magnetic saturation betweenabout 5 and about 90 EMU/g of metal oxide at approximately 300° K. and amagnetic squareness of less than 0.1, characteristic of asuperparamagnetic metal oxide crystal; (iii) being capable of retaininganions in solution, characteristic of paramagnetic metal oxyhydroxides;(iv) being capable of producing proton relaxivity values, R₁ and R₂,greater than or equal to about 10⁴ and 10⁵ M⁻¹ sec⁻¹, respectively,characteristic of sonicated superparamagnetic metal oxide crystalaggregates; and (v) capable of being biodegraded in a subject withinabout two weeks or less after administration, as evidenced by a returnof the proton relaxation rates of affected tissue of such subject topreadministration levels.
 2. A biodegradable superparamagnetic metaloxide comprising aggregates of individual biodegradablesuperparamagnetic hydrated metal oxide crystals associated with amacromolecular species, said metal oxide (i) having an individualcrystal diameter of about 500 angstroms or less and aggregates with anoverall mean diameter of about 4000 angstroms or less, as measured bylight scattering methods; (ii) having a magnetic saturation betweenabout 5 and about 90 EMU/g of metal oxide at approximately 300° K. and amagnetic squareness of less than 0.1, characteristic of asuperparamagnetic metal oxide crystal; (iii) being capable of retaininganions in solution; characteristic of paramagnetic metal oxyhydroxides;(iv) being capable of producing proton relaxivity values, R₁ and R₂,greater than or equal to about 10⁴ and 10⁵ M⁻¹ sec⁻¹, respectively,characteristic of sonicated superparamagnetic metal oxide crystalaggregates; about (v) capable of being biodegraded in a subject withinabout two weeks or less after administration, as evidenced by a returnof the proton relaxation rates of affected tissue of such subject topreadministration levels.
 3. The biodegradable superparamagnetic oxideof claim 1 or 2 in which said metal is selected from the groupconsisting of iron, cobalt, chromium, copper, manganese, molybdenum,nickel, and tungsten.
 4. The biodegradable superparamagnetic oxide ofclaim 1 or 2 in which said metal is iron.
 5. The biodegradablesuperparamagnetic oxide of claim 1 or 2 in which said metal oxide isselected from the group consisting of aggregates having an overall meandiameter less than about 3000, 2000, 1000, and 500 angstroms.
 6. Thebiodegradable superparamagnetic metal oxide of claim 2 in which saidmacromolecular species has a molecular weight of about 1 to about 250kilodaltons.
 7. The biodegradable superparamagnetic metal oxide of claim2 in which said macromolecular species is a carbohydrate.
 8. Thebiodegradable superparamagnetic metal oxide of claim 2 in which saidmacromolecular species is dextran.
 9. The biodegradablesuperparamagnetic metal oxide of claim 2 in which said macromolecularspecies and said metal are present in a weight ratio of about 0.01 toabout
 10. 10. The biodegradable superparamagnetic metal oxide of claim 2in which said macromolecular species and said metal are present in aweight ratio of about 0.01 to about 0.2.
 11. A sterilizablebiodegradable superparamagnetic fluid which comprises:(a) abiodegradable superparamagnetic metal oxide comprising aggregates ofindividual biodegradable superparamagnetic hydrated metal oxidecrystals, said metal oxide (i) having an individual crystal diameter ofabout 500 angstroms or less and aggregates with an overall mean diameterof about 4000 angstroms or less, as measured by light scatteringmethods; (ii) having a magnetic saturation between about 5 and about 90EMU/g of metal oxide at approximately 300° K. and a magnetic squarenessof less than 0.1, characteristic of a superparamagnetic metal oxidecrystal; (iii) being capable of retaining anions in solution,characteristic of paramagnetic metal oxyhydroxides; (iv) being capableof producing proton relaxivity values, R₁ and R₂, greater than or equalto about 10⁴ and 10⁵ M³¹ 1 sec⁻¹, respectively, characteristic ofsonicated superparamagnetic metal oxide crystal aggregates; and (v)capable of being biodegraded in a subject within about two weeks or lessafter administration, as evidenced by a return of the proton relaxationrates of affected tissue of such subject to preadministration levels;and (b) a physiologically acceptable medium.
 12. A sterilizablebiodegradable superparamagnetic fluid which comprises:(a) abiodegradable superparamagnetic metal oxide comprising aggregates ofindividual biodegradable superparamagnetic hydrated metal oxide crystalsassociated with a macromolecular species, said metal oxide (i) having anindividual crystal diameter of about 500 angstroms or less andaggregates with an overall mean diameter of about 4000 angstroms orless, as measured by light scattering methods; (ii) having a magneticsaturation between about 5 and about 90 EMU/g of metal oxide atapproximately 300° K. and a magnetic squareness of less than 0.1,characteristic of a superparamagnetic metal oxide crystal; (iii) beingcapable of retaining anions in solutions, characteristic of paramagneticmetal oxyhydroxides: (iv) being capable of producing proton relaxivityvalues, R₁ and R₂, greater than or equal to about 10⁴ and 10⁵ M³¹ 1sec³¹ 1, respectively, characteristic of sonicated superparamagneticmetal oxide crystal aggregates; and (v) capable of being biodegraded ina subject within about two weeks or less after administration, asevidenced by a return of the proton relaxation rates of affected tissueof such subject to preadministration levels; and (b) a physiologicallyacceptable medium.
 13. The sterilizable biodegradable superparamagneticfluid of claim 11 or 12 in which said medium comprises an aqueoussolution of a polyfunctional organic molecule selected from the groupconsisting of polyphosphates, polyphosphinates, polyphosphonates,polysulfinates, polysulfonates, and polycarboxylates.
 14. Thesterilizable biodegradable superparamagnetic fluid of claim 11 or 12 inwhich said medium comprises an aqueous buffer selected from the groupconsisting of ethylenediamine polyacetate, citrate, tartrate, succinate,and maleatic buffers.
 15. The sterilizable biodegradable fluid of claim11 or 12 in which said medium comprises an aqueous buffer selected fromthe group consisting of sodium, potassium, and ammonium citrate buffers.16. The sterilizable biodegradable fluid of claim 11 or 12 in which saidmedium contains added salt selected froam the group consisting of sodiumchloride, potassium chloride, sodium iodide, and potassium iodide. 17.The sterilizable biodegradable fluid of claim 11 or 12 in which saidmetal is iron.
 18. An autoclavable biodegradable superparamagnetic fluidwhich comprises:(a) a biodegradable superparamagnetic iron oxidecomprising aggregates of individual biodegradable superparamagnetichydrated iron oxide crystals associated with dextran, said meal oxide(i) having an individual crystal diameter of about 500 angstroms or lessand aggregates with an overall mean diameter of about 4000 angstroms orless, as measured by light scattering methods; (ii) having a magneticsaturation between about 5 and about 90 EMU/g of iron oxide atapproximately 300° K. and a magnetic squareness of less than 0.1,characteristic of a superparamagnetic iron oxide crystal; (iii) beingcapable of retaining anions in solutions, characteristic of paramagneticiron oxyhydroxides; (iv) being capable of producing proton relaxivityvalues, R₁ and R₂, greater than or equal to about 10⁴ and 10⁵ M⁻¹ sec³¹1, respectively, characteristic of sonicated superparamagnetic ironoxide crystal aggregates; and (v) capable of being biodegraded in asubject within about two weeks or less after administration, asevidenced by a return of the proton relaxation rates of affected tissueof such subject to preadministration levels; and (b) an aqueous citratebuffer.
 19. The composition of claim 1, 2, 11, 12, or 18 which isfurther characterized as being hypotonic.
 20. The composition of claim1, 2, 11, 12, or 18 which is further characterized as being isotonic.21. A magnetic resonance imaging contrast agent in a physiologicallyacceptable medium, which contrast agent (i) comprises a biodegradablesuperparamagnetic metal oxide, characterized as being metabolized orexcreted by a subject within 30 days or less after administration, and(ii) has a blood half-life in the rat of at least about 17 minutes at adose of about 2 mg per kg of rat.
 22. The contrast agent of claim 21 inwhich said metal oxide is biodegraded in such subject within about twoweeks or less after administration, as evidenced by a return of theproton relaxation rates of affected tissue to pre-administration levels.23. The contrast agent of claim 21 in which said metal oxide isbiodegraded in such subject within about one week or less afteradministration, as evidenced by a return of the proton relaxation ratesof affected tissue to pre-administration levels.
 24. The contrast agentof claim 21, 22 or 23 in which said metal oxide is associated with amacromolecular species.
 25. The contrast agent of claim 24 in which saidmacromolecular species is a polysaccharide or mixtures thereof.
 26. Thecontrast agent of claim 21, 22 or 23 in which said metal is iron. 27.The biodegradable superparamagnetic metal oxide of claim 2 in which saidmacromolecular species is a polysaccharide or mixtures thereof.
 28. Thebiodegradable superparamagnetic metal oxide of claim 1, 2, 11 or 12 inwhich the individual crystal diameter is about 100 angstroms or less andthe overall mean diameter of the aggregates is about 500 angstroms orless, as measured by light scattering methods.
 29. The contrast agent ofclaim 24 which is further characterized as being capable of exerting abrightening effect upon administration to an animal or human subject.30. The contrast agent of claim 24 which is further characterized asbeing capable of exerting a darkening effect upon administration to ananimal or human subject.
 31. The contrast agent of claim 24 which isfurther characterized as being capable of exerting a contrast effectthat is a combination of a brightening effect and a darkening effectupon administration to an animal or human subject.
 32. The contrastagent of claim 21, 22 or 23 in which said blood half-life in the rate isat least about 50 minutes at a dose of 2 mg per kg of rat.
 33. Thecontrast agent of claim 21, 22, 24 in which said metal is iron.
 34. Thecontrast agent of claim 24 in which said metal oxide is comprised of oneor more biodegradable superparamagnetic metal oxide crystals, saidcrystals having an overall mean diameter of about 100 angstroms or less.35. The contrast agent of claim 24 in which said metal oxide iscomprised of aggregates of metal oxide crystals, said aggregates havingan overall mean diameter of about 3000 angstroms or less.
 36. Thecontrast agent of claim 24 in which said metal oxide is comprised ofaggregates of metal oxide crystals, said aggregates having an overallmean diameter of about 2000 angstroms or less.
 37. The contrast agent ofclaim 24 in which said metal oxide is comprised of aggregates of metaloxide crystals, said aggregates having an overall mean diameter of about1000 angstroms or less.
 38. The contrast agent of claim 24 in which saidmetal oxide is comprised of aggregates of metal oxide crystals, saidaggregates having an overall mean diameter of about 500 angstroms orless.
 39. The contrast agent of claim 21, 22 or 23 in which said mealoxide is comprised of one or more biodegradable superparamagnetic mealoxide crystals, each crystal having a diameter of about 100 angstroms orless and the largest aggregate of metal oxide crystals having an overallmean diameter not exceeding about 500 angstroms.
 40. The contrast agentof claim 24 in which said metal oxide is comprised of one or morebiodegradable superparamagnetic metal oxide crystals, each crystalhaving a diameter of about 100 angstroms or less and the largestaggregates of metal oxide crystals having an overall mean diameter notexceeding about 500 angstroms.
 41. The contrast agent of claim 25 inwhich said metal is iron.
 42. The contrast agent of claim 24 in whichsaid blood half-life in the rat is at least about 50 minutes at a doseof 2 mg per kg of rat.
 43. The contrast agent of claim 25 in which saidblood half-life in the rat is at least about 50 minutes at a dose of 2mg per kg of rat.